Human serum albumin (hereunder also referred to as “HSA”) is a principal protein component present in the plasma, consists of a single chain polypeptide comprising 585 amino acid residues and has a molecular weight equal to about 66,000 Dalton (see Minghetti, P. P. et al. (1986), Molecular structure of the human albumin gene is revealed by nucleotide sequence within 11–22 of chromosome 4. J. Biol. Chem. 261, pp. 6747–6757). It has been known that the principal roles of HSA are not only to maintain the normal osmotic pressure of the blood, but also to bind with a variety of substances such as calcium ions, fatty acids, bilirubin, tryptophan and drugs possibly present in the blood, thereby playing a role of a carrier for transporting these substances. Purified HSA has been used in, for instance, the postoperative treatment after surgical operations and the treatment of hypoalbuminemia caused due to the loss of albumin such as hemorrhagic shock, burn and nephrotic syndromes.
Conventionally, HSA has been prepared by subjecting the human plasma to the low temperature ethanol-fractionation method of Cone or any method similar thereto to give HSA-containing fractions (HSA is fractionated in the fraction V) and then purifying the fraction through the use of a variety of purification techniques. Moreover, there has recently been developed a method in which the human plasma is not used as a raw material, for instance, a technique for producing human serum albumin using yeast, Escherichia coli or Bacillus subtilis cells, while making use of the gene recombination technique.
These gene recombination techniques are detailed in (1) Production of recombinant Human Serum Albumin from Saccharomyces cerevisiae; Quirk, R. et al. Biotechnology and Applied Biochemistry, 1989, 11: 273–287, (2) Secretory Expression of the Human Serum Albumin Gene in the Yeast, Saccharomyces cerevisiae; Ken Okabayashi et al. J. Biochemistry, 1991, 110: 103–110, (3) Yeast Systems for the Commercial Production of Heterologous Proteins; Richard G. Buckholz and Martin A. G. Gleeson, Bio/Technology, 1991, 9: 1067–1072 for the yeast, (4) Construction of DNA sequences and their use for microbial production of proteins, in particular, human serum albumin; Lawn, R. M. European Patent Publication No. 0073646A (1983), (5) Synthesis and Purification of mature human serum albumin from E. coli; Latta, L. et al. Biotechnique, 1897, 5: 1309–1314 for the Escherichia coli (E. coli), (6) Secretion of human serum albumin from Bacillus subtilis; Saunders, C. W. et al. J. Bacteriol. 1987, 169: 2917–2925 for the Bacillus subtilis. 
The methods for purifying the human serum albumin usable herein in general include those currently used in the protein chemistry such as a salting out method, an ultrafiltration method, an isoelectric precipitation method, an electrophoresis method, an ion-exchange chromatography technique, a gel filtration chromatography technique and/or an affinity chromatography technique. Indeed, the human serum albumin-containing fraction includes various kinds of contaminants originated from, for instance, biological tissues, cells and blood and therefore, the human serum albumin has been purified by a complicated combination of the foregoing methods.
In the industrial production of human serum albumin, it is inevitable to treat the same under various conditions different from environmental conditions observed in the human body and accordingly, multimers of human serum albumin are formed. There has not yet been known any such a report that these multimers adversely affect the human body in the clinical application of human serum albumin, but there is such a suspicion that these multimers may develop a novel antigenicity. For this reason, an upper limit in the contamination with these multimers is prescribed in the standardization test of “human serum albumin” as a pharmaceutical agent from the viewpoint of the safety thereof as a medicine and therefore, it becomes an important problem, in the production of a pharmaceutical preparation containing the same, to substantially reduce the content of such multimers in the preparation.
Two or more molecules of human serum albumin are linked with one another to form such a multimer thereof, the isoelectric point and chemical characteristics of the latter are correspondingly quite similar to those observed for the monomer and therefore, it is very difficult to separate the multimers from the monomers according to the purification methods employed in the usual production process. For this reason, the multimers have been removed, in the conventional techniques, by a combination of several kinds of purification methods selected from the group consisting of gel filtration chromatography, ion-exchange chromatography, affinity chromatography, isoelectric fractionation, ammonium sulfate fractionation and ethanol fractionation techniques (see, TOKUHYO Hei 11-509525 (International Patent Publication WO96/37515) and Japanese Patent No. 2,926,722 (registered on Heisei 11 (1999), May 14)).
If a plurality of purification methods is used in combination, the final rate of recovery is the product of those achieved in the purification steps used and therefore, the resulting productivity is significantly reduced in most cases. Accordingly, there has been desired for the development of a method, which can reduce the number of purification steps as low as possible, permits the effective removal of multimers of human serum albumin and allows the recovery of monomers thereof at a high yield, from the industrial standpoint.